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Comparing FLG Genes of Atopic Dermatitis Phenotype with Reference Genes
Introduction and Background
Literature Review
There are many skin diseases, irritations, lesions, and inflammations. Two particular diseases of interest are atopic dermatitis (AD) and psoriasis. To many observers, these conditions appear similar and/or synonymous. It is speculated by some researchers and clinicians that the diseases may have a common etiology. One may also argue that upon closer examination these diseases or respective spectrums are fundamentally very different in morphology.
However, AD and psoriasis do share some common inflammation mechanisms, and as it turns out - there is a discrete overlap in etiology. Among the bio-medical scientific community, there are several genes already known to be associated with psoriasis. A few notable papers will be mentioned throughout this brief introduction. At the submission of this project, there is one protein of interest for AD – filaggrin; this protein is coded for by the FLG gene. There is a direct correlation between diminished filaggrin production and atopic dermatitis. This association was demonstrated by both Smith and Barker and their respective teams (2006, 2007). To reiterate, both AD and psoriasis patients are likely predisposed to their respective diseases by a deficiency in filaggrin protein. Albeit, there are other cofactors and many dermal agitators. Yet, there is a clear correlation between filaggrin deficiency and atopic dermatitis from the literature. For brevity, most of this project’s research will focus on AD. They are often considered synonymous with eczema. This brings us no closer to the answer of what exactly any of these diseases entail. In English, translated very literally from the Greek, eczema simply means, “To boil over (Dobson, 1976).”
To that regard, some physicians are concerned that even eczema is too vague of a description of several dermatoses (Boss, 1998). In clinical practice, they do form a similar group - at least from a morphological point of view (1998). That is, they have ill-defined borders, redness or erythema, vesiculations, lichenifications, plaques, scaling, and sometimes even nodules (1998). They characteristically demonstrate epidermo-dermatitis, a mononuclear (lymphohistiocytic) infiltrate, and accumulation of lymphocytes (1998). Of note, there is an increase in secretions of pro-inflammatory cytokines like interleukin 1β (IL-1β), IL-6, tumor necrosis factor a, (TNFα) and IL-8 (1998).
It can reasonably be argued that several designated examples of eczema are characterized as atopic dermatitis. Yes, many researchers consider these terms synonymous. However, for the sake of saving explanation time and space, this project recognizes AD as types of eczema. Again, atopic dermatitis itself may be generally regarded as a collection of related skin diseases. From these two paradigms, I deduce that the spectrum of eczemas are broader than that of AD.
AD is characterized by severe pruritus, chronically relapsing course, and a distinctive distribution of eczematous skin lesions (Breuer, Kapp, and Werfel, 2001). There is often a personal or family history of atopic diseases (2001). Furthermore, a mononuclear infiltrate, dominated by CD4+ T-helper cells, is found in the dermis. The condition is induced or exacerbated by several internal and external factors (2001). As alluded to above, one such factor is inadequate filaggrin production.
“Filaggrin is essential for the cell compaction process that precedes chemical crosslinking and skin barrier formation. (Barker et al, 2007).” For Homo sapiens (human), this gene is located in the epidermal differentiation complex of 1q21 (Mischke, Korge, Marenholz, Volz, & Ziegler 1996).
For Mus musculus (mouse), it is on chromosome 3 at the locus: 3 F2.1; 3 40.16 cM (Rothnagel et al., 1994). Null mutations in this gene were characterized in humans1 by Smith et al; his team designated R501X and 2282del4 (2006). There is also reference data available through NCBI: c.3321delA for humans; complete cds for FLG gene of flaky tail mouse.
In summary, inadequate barrier formation factors preceded by inherently deficient filaggrin production are likely predisposing conditions for atopic dermatitis (Barker et al, 2007). Thus, as noted by both Barker and Smith teams, the null mutations in the corresponding FLG gene are of significant mention. Barker’s theory suggests that the absence of these barrier factors leads to skin that is inherently more permeable (2007). Therefore, allergens and irritants are freer to enter the skin. Scratching produces more lesions, irritations, reservoir for infection, and further increases permeability (2007).
1The Literature Review aspect of this manuscript will focus on the association between atopic dermatitis and filaggrin production in humans. Comparison studies will only briefly be reviewed for mice in the next section. For this project, we will mainly be using the Mus musculus sequence data from NCBI. Therefore, please note that all references in this project are for human studies unless specially mentioned otherwise.
Literature Update
Atopic dermatitis arises from a variety of gene aberrations, protein malformations, and environmental conditions acting in concert (Tsuji, 2018). OVOL1-OVOL2 should also be considered as part of this medley as OVOL1 has a relationship with filaggrin (Tsuji, 2018). Additionally, Engebretsen’s team demonstrated that natural moisturizing factors (NMF) were reduced in patients with mutations in the FLG gene (2018). This method gives us one more means to ascertain filaggrin’s role in atopic dermatitis.
Finally, sensitive skin and disease severity are noted in an AD cohort with FLG aberrations (Yatagai et al., 2018). Interestingly, this is not directly correlated with barrier impairment for this particular study (Yatagai et al, 2018).
Project Motivation
There are a great number of people afflicted with atopic dermatitis, the author of this essay included. At the submission of this project, there is no cure for AD. The exact cause of these diseases remains unknown. However, genetic studies have revealed at least one plausible contributor or co-factor to disease onset. Further study must be done in correlating and corroborating the data related to this gene. In other words, the connection between filaggrin deficiency and AD in human populations has been well studied. This association has also been demonstrated in mice: (Kezic et al, 2012); (Moniaga et al, 2010); (Fallon, 2009). It is the goal of this project to pool and synthesize these findings. This is with special attention to FLG gene variants. The FLG genes and protein translation sequences of at least 4 donors were compared. My research demonstrated that the gene is highly conserved among the two mammalian populations. I was also able to authenticate that there are deletions in JN184346.1 and FJ824603. Furthermore, these aberrations do cause appreciable protein translation changes.
Goals
This project was very small in scope. I achieved three main tasks and compared 4 distinct FLG genes as specified in the previous section. Two FLG genes were from psoriasis and/or atopic dermatitis (+) positive donors. These donors have clinically diagnosed psoriasis and/or AD phenotypes. The and/or designation is given because it is harder to ascertain whether the flaky tail mouse has AD or psoriasis. Two genes were atopic dermatitis and psoriasis (-) negative, or non-affected. The latter two were the reference FLG genes from NCBI's database.
These next three tasks outline the basic project constituents or components.
Task 1. We performed the following gene comparisons:
FLG gene in atopic dermatitis + and – human populations;
FLG gene in atopic dermatitis + and – mouse populations.
Task 2. We performed the following cross-species comparisons:
FLG reference genes in human and mouse to each other;
FLG reference gene in human to flaky tail mouse (dermatitis +);
FLG dermatitis variants in human and mouse to each other.
Task 3. We performed the same comparisons on the translated protein sequences.
Two great resources for bioinformatics studies are The National Center for Biotechnology Information (NCBI) and the University of California Santa Cruz (UCSC). Both the NCBI and the UCSC genome browsers allow us to look at the reference sequences. This was done with the FLG genes of the two species of interest. We have also made use of Qiagen’s CLC Sequence Viewer 8.0.0 and ClusalW for viewing data in this project.
Results
Please, note that tasks 1-3 are repeated and re-arranged as necessary.
Task 1 - Homo sapiens
We should begin by comparing the relevant region of the reference FLG gene
(NG_016190.1) with the end of the dermatitis FLG gene (JN184346.1) in humans. This can be done with NCBI’s BLAST or a multiple sequence alignment by ClustalW. In this example, we used the latter.
Figure 1. DNA sequence comparison showing adenine deletion.
The results in Figure 1 were provided by Arif Harmanci. This alignment serves as a great reference point. These nucleotide pairings demonstrate the deletion of an adenine at the 3321 position. These results proved difficult to repeat and/or elicit using NCBI BLAST or ClustalW.
Instead, this alignment has been reproduced with Qiagen’s CLC Sequence viewer 8.0.0. The color scheme and numbering used by this software makes visualizing the deletion less arduous.
Figure 2. Qiagen’s CLC Sequence viewer output.
Again, color coding, base numbering, and the superior alignment of Qiagen’s software algorithm make finding this single nucleotide adenine deletion easy to identify. It is at the position 3321 on the reference gene (top row, 5th group).
Accordingly, the second row changes by a decrease of 1 on the nucleotide counting. Deletions like these can alter the reading frame and ultimately protein translation. Thus, it is imperative that we compare the relevant portions of the reference protein with the affected dermatitis protein.
Task 3 - Homo sapiens
Figure 3. Protein alignment at divergent point.
Figure 4. Protein sequence alignment from Qiagen's CLC sequence viewer 8.0.0.
The reference protein continues on at position 1109 as demonstrated in Figure 4. As one would expect, there is a frame-shift mutation at this loci; the 1109 amino acid position for the c.3321delA (JN184346.1). Again, this location is being numbered from the reference protein sequence. If the view is expanded to include the next group, we can discern that the software is still attempting alignment. You may notice that it places EGLD somewhat distant from 1109 and almost seemingly random.
Figure 5. Expanded view of protein alignment from divergent point at 1109. [Please, see figure 4].
Task 1 - Mus musculus
Similarly, we find aberrations if we juxtapose the mouse data. There are single nucleotide polymorphisms (SNPs) in the gene sequence at 1263 and 1634. Obviously, these changes are in the flaky tail mouse gene (FJ824603.1). For these two respective locations: guanine is turned to cytosine; thymine is turned to cytosine. There is more sequence date available for the flaky tail mouse in this comparison. The reference mouse’s gene (AY094988.1) coding sequence dropped off at 1660 and resumed at 1661 for termination.
Figure 6. SNP of flaky tail mouse at 1263.
… (approximately 300bp non-variant nucleotide groups omitted)
Figure 7. SNP of flaky tail mouse at 1634 and break from reference sequence at 1661.
Task 3 - Mus musculus
If we take a look at the mouse protein sequence, we see that a valine turns to an alanine. This is what we would expect from GTA (GUA) → GCA at the 445 amino acid position [1634 nucleotide position]. Accordingly, our coding sequence also ends at 553 for our reference gene.
Figure 8. Flaky tail amino acid substitution at position 445.
Task 2 - Cross-species gene comparison
These acronyms were created for use in the following tables of this essay:
Table 1. Acronyms, NCBI reference numbers, and short definitions.
Table 2. Cross-species gene comparison statistics. [Summary Omitted. Click on buttons for full reports].
Task 3 - Cross-species protein comparison
Table 3. Cross-species protein comparison statistics. [Summary Omitted. Click on buttons for full reports].
These results are the most pertinent combinations of these 4 coding sequence and their expressed proteins interspecies. Notably: The comparison for the cds (coding sequences) yielded about 72% identity match. However, the translated protein was only 31%-37% similar. The greatest similarity was between the human and mouse reference (non-diseased) sequences.
Methods and Data Sets
From the literature review, this project reaffirms that a deficiency in filaggrin can make an organism more susceptible to dermal conditions. This association has not only been studied in humans, but in mice as well. However, this project’s research focus was on the comparison of the corresponding and homologous FLG genes of two species not filaggrin expression.
Methods Summary
The data analyzed was for subjects both with and without atopic dermatitis. 1st - A brief literature review was conducted. 2nd - NCBI’s BLAST feature was utilized to locate and/or confirm the respective reference(s). BLAST was also used to compare the FLG gene from one species to another as specified in the Goals section of this project. 3rd - Qiagen’s CLC Sequence Viewer 8.0.0 was used where BLAST data was more difficult to interpret. For those unaware, BLAST is, “A new [sic] approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. (Altschul et al. 1990, pp 403).” 4th – A second smaller literature review was conducted for 2018 data to insure relevance of findings.
Methods for literature review
Initially, Google Scholar was queried with the search terms atopic dermatitis and gene. After reviewing some of the literature, the candidate gene FLG and its associated filaggrin protein were queried separately via Google Scholar. It was noted that some studies had been conducted on mice and dogs; both species share a homologous FLG gene. Queries were also done to include these two non-human mammals. Several articles were reviewed and selected for use as background sources for this project. This information was great in elicited our proposed thesis; it was also a great starting point for bioinformatics research. Specifically, several diseased phenotypes, SNPs, and nucleotide deletions were named in these papers.
However, some of the articles were a little dated by modern bioinformatics research standards. Therefore, on 11-21-2018 a more thorough literature review was initiated, the Pubmed database was queried with the keywords FLG and filaggrin. This query was paired with the Mesh term, “Dermatitis, Atopic.” The AND operand was utilized to ensure that both keywords and the Mesh term all appeared in the returned literature. The exact query was as follows: (FLG) AND (filaggrin) AND "Dermatitis, Atopic"[Mesh]. 215 articles were returned in the initial search. Articles were reviewed based on title and abstract for relevant themes and topics. The intent of this research was to investigate the association between the FLG gene, filaggrin protein, and atopic dermatitis. The articles were arranged so that I could view the most recent material on top of the returned search query. Only articles published in the year 2018 were considered for this literature update. This limited the number of supplemental Literature Update articles to 3.
Methods for BLAST
From NCBI, we obtained 4 nucleotide and 4 protein sequences. The sequence data was copied in the FASTA format. This information was placed in text files named by their NCBI reference IDs: NG_016190.1; JN184346.1; AY094988.1; FJ824603.1. The corresponding protein sequences were given (p-) prefixes: p-NG_016190.1; p-JN184346.1; p-AY094988.1; p-FJ824603.1. Comparisons were made intraspecies and interspecies as described in both the Goals and Results sections of this manuscript. The three primary software tools used were: NCBI Nucleotide BLAST2; NCBI Protein BLAST3; Qiagen CLC Sequence Viewer4. The settings for the two BLAST’s were set to somewhat similar and the box for, “Align two or more sequences,” was checked. The FASTA sequence data was pasted into the two respective boxes.
The 64-bit.exe of Qiagen’s CLC Sequence Viewer 8.0.0 was downloaded. The program was installed and executed on a HP ProBook 6470b running a 64-bit version of Windows 7. The specifications of the laptop were: Core i5 3210M; 500 gigabyte hard drive; 4GB RAM. The settings for the viewer were not changed. The saved sequence data text files were imported into the software. Two files were selected after they were imported into the CLC Viewer. Ctrl+Shift+A was pressed to compare the two sequences; this hotkey combination initiated the alignment function. Alternatively, you can navigate from the top menu: toolbox --> alignments and trees --> create alignment. Screenshots were taken of pertinent results.
2https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
4https://www.qiagenbioinformatics.com/products/clc-sequence-viewer-direct-download/
Discussion
The original focus of this project was to validate a few studies demonstrating the association between atopic dermatitis and diminished filaggrin protein production. This is partly because this association is at the very least correlated. The conclusions of two of the studies mentioned in the Literature Review affirm this correlation: (Barker, 2007); (Smith, 2006). I set out to review the methods they employed and their data sets to confirm this association. DNA samples and sequences were not readily made available by the primary researchers. Fortunately, NCBI did have FLG reference data for human and mouse: c.3321delA for human; the complete FLG cds for the flaky tail mouse. The focus of this project deviated from validating previous research. Thus, the stated thesis of this project was updated to analysis of four FLG genes and their corresponding amino acid protein sequences. There were several Nucleotide BLASTs performed on NCBI. Intraspecies - these studies did allow us to verify the findings of some previously acknowledged researchers. Again, it was my hope to simply visualize the deletions against the reference genomes.
Shortcomings
One of the shortcomings of this current study is that not many affected phenotypes were cataloged or publically available. This would have allowed for a succinct meta-analysis. Thus, additional searches need to be made to locate sequence data of confirmed atopic dermatitis donors. Sequence data for R501X and 2282del4 are available, and a greater effort should be made to acquire this information. It is very likely that the authors of several publications will need to be contacted directly. It is my hope that they would be able to provide the sequence data of more AD donors for the FLG gene.
Once obtained, the data from the AD donors could be compared to the non-atopic dermatitis reference sequence. These samples could also be compared to c.3321delA and (JN184346.1) from NCBI. Of note, the DNA sequence data for the FLG c.3321delA variant is from a patient with psoriasis not atopic dermatitis. As mentioned in the literature review, AD and psoriasis do have some etiological overlap. Variant c.3321delA has been demonstrated in dermatitis patients (Meng et al, 2014). Therefore, the sequence data for a psoriasis patient was used as it was readily available through NCBI.
Future Research
In the future, I would also like to expand this research to other eczemas, and the spectrum of psoriasis. Furthermore, given the interest in the filaggrin protein and the FLG gene correlation, additional information about FLG can be examined. Research based on its gene and/or protein family may also be conducted. As a primer to that, filaggrin is known to be part of the S100 proteins. Interestingly, “Of the 24 human S100 genes, 19 (S100 proteins, group A) are located within chromosome 1q21 (Marenholz, Heizmann, & Fritz, 2004).”
Data from domesticated dogs should be included in a future study. The patient sample size can potentially be very large. This is because concerned pet owners often bring atopic dermatitis affected dogs to the veterinarian. Initially, we did set out to include limited canine data in this study. In the literature, there are many researchers looking for a connection between dogs and FLG production: (Kanda, 2013); (Barros Roque et al, 2009). However, no FLG sequence data was available on NCBI. For Canis lupus familiaris, it has been noted the gene is somewhere on Chromosome 17 (Suriyaphol et al., 2011). To that regard, the dog chromosome 17 is a relatively large data file. Furthermore, no dermatitis affected sequences are readily available. I was not able to elicit the FLG gene for a dog at the time of this study.
References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Barker, J. N. W. N., Palmer, C. N. A., Zhao, Y., Liao, H., Hull, P. R., Lee, S. P., … Mclean, W.
H. I. (2007). Null Mutations in the Filaggrin Gene ( FLG ) Determine Major Susceptibility to Early-Onset Atopic Dermatitis that Persists into Adulthood. Journal of Investigative Dermatology, 127(3), 564–567. https://doi.org/10.1038/sj.jid.5700587
Barros Roque, J., O’Leary, C. A., Kyaw-Tanner, M., Latter, M., Mason, K., Shipstone, M., …
Duffy, D. L. (2009). Haplotype sharing excludes canine orthologous Filaggrin locus in atopy in West Highland White Terriers. Animal Genetics, 40(5), 793–794. https://doi.org/10.1111/j.1365-2052.2009.01915.x
Boss, J.D. (1998). Eczema. Encyclopedia of Immunology, 786-788. 2nd Edition. Elsevier, ltd. https://doi.org/10.1006/rwei.1999.0207
Breuer, K., Kapp, a, & Werfel, T. (2001). Bacterial infections and atopic dermatitis. Allergy, 56(11), 1034–1041. https://doi.org/10.1034/j.1398-9995.2001.00146.x
Dobson, R.L. (1976). Diagnosis and Treatment of Eczema. JAMA. ;235(20):2228–2229.
doi:10.1001/jama.1976.03260460048027
Engebretsen, K. , Bandier, J. , Kezic, S. , Riethmüller, C. , Heegaard, N. , Carlsen, B. ,
Linneberg, A. , Johansen, J. and Thyssen, J. (2018), Concentration of filaggrin monomers, its metabolites and corneocyte surface texture in individuals with a history of atopic dermatitis and controls. J Eur Acad Dermatol Venereol, 32: 796-804. doi:10.1111/jdv.14801
Fallon, P. G., Sasaki, T., Sandilands, A., Campbell, L. E., Saunders, S. P., Mangan, N. E.,
Callanan, J. J., Kawasaki, H., Shiohama, A., Kubo, A., Sundberg, J. P., Presland, R. B., Fleckman, P., Shimizu, N., Kudoh, J., Irvine, A. D., Amagai, M., … McLean, W. H. (2009). A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming. Nature genetics, 41(5), 602-8.
Kanda, S., Sasaki, T., Shiohama, A., Nishifuji, K., Amagai, M., Iwasaki, T., & Kudoh, J. (2013). Characterization of canine filaggrin: Gene structure and protein expression in dog skin. Veterinary Dermatology, 24(1), 25–32. https://doi.org/10.1111/j.1365-3164.2012.01105.x
Kezic, S., O’Regan, G. M., Lutter, R., Jakasa, I., Koster, E. S., Saunders, S., … Irvine, A. D. (2012). Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency. Journal of Allergy and Clinical Immunology, 129(4), 1031–1040. https://doi.org/10.1016/j.jaci.2011.12.989
Marenholz, I., Heizmann, C. W., & Fritz, G. (2004). S100 proteins in mouse and man: From evolution to function and pathology (including an update of the nomenclature). Biochemical and Biophysical Research Communications, 322(4), 1111–1122. https://doi.org/10.1016/j.bbrc.2004.07.096
Meng, L., Wang, L., Tang, H., Tang, X., Jiang, X., Zhao, J., Gao, J., Li, B., Fu, X., Chen, Y.,
Yao, W., Zhan, W., Wu, B., Duan, D., Shen, C., Cheng, H., Zuo, X., Yang, S., Sun, L., … Zhang, X. (2014). Filaggrin gene mutation c.3321delA is associated with various clinical features of atopic dermatitis in the Chinese Han population. PloS one, 9(5), e98235. doi:10.1371/journal.pone.0098235
Mischke, D., Korge, B. P., Marenholz, I., Volz, A., & Ziegler, A. (1996). Genes Encoding Structural Proteins of Epidermal Cornification and S100 Calcium-Binding Proteins Form a Gene Complex (“Epidermal Differentiation Complex”) on Human Chromosome 1q21. Journal of Investigative Dermatology, 106(5), 989–992. https://doi.org/10.1111/1523-1747.ep12338501
Moniaga, C. S., Egawa, G., Kawasaki, H., Hara-Chikuma, M., Honda, T., Tanizaki, H., … Kabashima, K. (2010). Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract. American Journal of Pathology, 176(5), 2385–2393. https://doi.org/10.2353/ajpath.2010.090957
Rothnagel, J. A., Longley, M. A., Bundman, D. S., Naylor, S. L., Lalley, P. A., Jenkins, N. A., … Roop, D. R. (1994). Characterization of the Mouse Loricrin Gene: Linkage with Profilaggrin and the Flaky Tail and Soft Coat Mutant Loci on Chromosome 3. Genomics, 23(2), 450–456. https://doi.org/10.1006/GENO.1994.1522
Smith, F. J. D., Irvine, A. D., Terron-Kwiatkowski, A., Sandilands, A., Campbell, L. E., Zhao, Y., … McLean, W.
H. I. (2006). Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nature Genetics, 38(3), 337–342. https://doi.org/10.1038/ng1743
Suriyaphol, G., Suriyaphol P., Sarikaputi, M., Theerawatanasirikul, S., Sailasuta, A. (2011). Association of filaggrin (FLG) gene polymorphism with canine atopic dermatitis in small breed dogs. Thai Journal of Veterinary Medicine, 41(4), 509-517. http://www.thaiscience.info/Journals/Article/TJVM/10889456.pdf
Tsuji, G., Ito, T., Chiba, T. Mitoma, C., Nakahara, T., Uchi, H., Furue, M., (2018). The role of
the OVOL1-OVOL2 axis in normal and diseased human skin. Journal of Dermatological Science, Volume 90, Issue 3, Pages 227-231. doi: 10.1016/j.jdermsci.2018.02.005
Yatagai,T., Shimauchi, T., Yamaguchi, H., Sakabe, J., Aoshima, M., Ikeya, S., Tatsuno, K., Fujiyama, T., Ito, T., Ojima, T., Tokura, Y. (2018). Sensitive skin is highly frequent in
extrinsic atopic dermatitis and correlates with disease severity markers but not necessarily with skin barrier impairment. Journal of Dermatological Science, 89(1), 33-39. doi: 10.1016/j.jdermsci.2017.10.011
